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Modulation of abnormal synaptic transmission in hippocampal CA3 neurons of spontaneously epileptic rats (SERs) by levetiracetam
Brain Research Bulletin 86 (2011) 334–339
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Modulation of abnormal synaptic transmission in hippocampal CA3 neurons of spontaneously epileptic rats (SERs) by levetiracetam Ryosuke Hanaya a,∗ , Yoshihiro Kiura b , Tadao Serikawa c , Kaoru Kurisu b , Kazunori Arita a , Masashi Sasa d a
Department of Neurosurgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan Department of Neurosurgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan d Nagisa Clinic, Hirakata 573-1183, Japan b c
a r t i c l e
i n f o
Article history: Received 4 August 2011 Received in revised form 17 September 2011 Accepted 19 September 2011 Available online 25 September 2011 Keywords: Levetiracetam Spontaneously epileptic rat (SER) Hippocampal CA3 neurons Depolarization shift GABAergic transmission Sodium channel
a b s t r a c t Levetiracetam (LEV) inhibits partial refractory epilepsy in human, and both convulsive and absence-like seizures in the spontaneously epileptic rat (SER). Two-thirds of hippocampal CA3 neurons in SER show a long-lasting depolarization shift, with accompanying repetitive firing upon mossy fiber stimulation. This abnormal excitability is probably attributable to abnormalities in the L-type Ca2+ channels. We performed electrophysiological studies to elucidate the mechanism underlying the antiepileptic effects of LEV via intracellular recording from the hippocampal CA3 neurons in slice preparations of SER and non-epileptic Wistar rats. LEV (100 M) inhibited the depolarization shift with repetitive firing by mossy fiber stimulation (MFS), without affecting the first spike in SER CA3 neurons. At a higher dose (1 mM), LEV suppressed the first spike in all SER neurons (including the CA3 neurons which showed only a single action potential by MFS), while the single action potential of Wistar rat CA3 neurons remained unaffected. SER CA3 neurons with MFS-induced abnormal firing exhibited a higher number of repetitive spikes when a depolarization pulse was applied in the SER CA3 neurons. LEV (100 M, 1 mM) reduced the repetitive firing induced by a depolarization pulse applied without affecting Ca2+ spike in SER neurons. LEV is known not to bind glutamate and gamma-aminobutyric acid (GABA) receptors. These findings suggest that the therapeutic concentration of LEV inhibits abnormal firing of the CA3 neurons by modulating abnormal synaptic transmission and abnormal Na+ channels in SER. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Levetiracetam (LEV), a pyrrolidone derivative structurally related to piracetam [4], is effective to inhibit the seizures in kindled and genetic animals [5,18,21] and for the treatment of both primary generalized and partial epilepsy in humans [15,20]. LEV, interestingly, has no remarkable effects on acute maximal electroshock or pentylentetrazole-induced seizures [18]. Unlike the conventional antiepileptic drugs, LEV does not directly affect either Na+ channels, T-type Ca2+ channels [37], glutamate receptors [9], or gamma-aminobutyric acid (GABA) systems [3,24]. LEV has been found to inhibit N-type Ca2+ channels [22] and to specifically bind to synaptic vesicle protein 2A (SV2A) [23], which is presumably involved in regulating neurotransmitter release [2].
∗ Corresponding author at: Department of Neurosurgery, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. Tel.: +81 99 275 5375; fax: +81 99 265 4041. E-mail address: [email protected] (R. Hanaya). 0361-9230/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2011.09.015
Our previous studies have demonstrated that LEV is effective in inhibiting both tonic convulsion and absence-like seizures in spontaneously epileptic rats (SERs: zi/zi, tm/tm) [12,35]. SER is a double mutant obtained by mating heterozygous tremor rats (tm/+), a mutant found in an inbred colony of Kyoto-Wistar rats [33], with homozygous zitter rats (zi/zi) originated from a Sprague-Dawley colony [26]. After 6 weeks of age, SERs spontaneously begin to show tonic convulsion and absence-like seizures characterized by respectively low voltage fast waves and 5–7 Hz spike-wave-like seizures in cortical and hippocampal EEG [27,28]. The profiles of conventional antiepileptic drugs for SER parallel those in human epilepsy [28]. A single stimulation of the mossy fibers (MF) induces a long-lasting (60–200 ms) depolarization shift, accompanied by repetitive firing and slow after-hyperpolarization in two thirds of the hippocampal CA3 neurons in SER [10]. Enhancement of intracellular Ca2+ levels with the stimulation of MF has been observed in the hippocampal CA3 neurons of SER, compared with normal rats [1]. This abnormal excitability would induce abnormalities in the L-type Ca2+ channels [25,34]. The MF-hippocampal CA3 neuron system of SER is one of the useful targets for elucidating the
R. Hanaya et al. / Brain Research Bulletin 86 (2011) 334–339
mechanism underlying the antiepileptic action of drugs [8,7]. We performed electrophysiological studies to elucidate the antiepileptic mechanism of LEV using SER hippocampal slices. 2. Materials and methods 2.1. Experimental animals SERs were bred in a specific pathogen-free barriered facility in the Institute of Laboratory Animals, Kyoto University, and were kept individually in shoebox type cages on laminar flow shelves in a conventional room throughout the experiment. Animal feed pellets (F-2, Funabashi Farm, Chiba, Japan) and drinking water were given to the animals ad libitum. The room temperature and relative humidity were maintained at 22–26 ◦ C and 50–65%, respectively. We used 29 mature (age: 10–16 weeks) SERs of either sex that exhibited convulsive seizures, and 10 age-matched Wistar rats, one of the parent strains of SERs, were used as controls. 2.2. Intracellular recording in hippocampal slices After deep anesthesia with ether, the brain of rodents was removed and rapidly placed in cold oxygenated Ca2+ -free medium (in mM: 113 NaCl, 3 KCl, 1 NaH2 PO4 , 25 NaHCO3 , 5 MgCl2 , 11 glucose), and hippocampal slices were cut at a thickness of 400 m with a microslicer (DTK-1000, Dosaka EM, Kyoto, Japan). After incubation for 1–2 h in artificial cerebrospinal fluid (ACSF; in mM: 113 NaCl, 3 KCl, 1 NaH2 PO4 , 25 NaHCO3 , 2 CaCl2 , 1 MgCl2 , 11 glucose; pH 7.2) at 34 ◦ C, the slices were placed into the recording chamber (volume: 0.7 cm3 ). ACSF continuously bubbled with a mixture of 95% O2 and 5% CO2 was perfused over the slice at a rate of 1.5–2.0 ml/min at room temperature. A bipolar stimulating electrode was placed in the granule cell layer of the dentate gyrus to provide a stimulus (0.1 ms-duration and 10–25 V) to the MF every 5 s. The MF stimulation (MFS) raised the threshold by 0.5 V, and held the intensity at 0.5 V higher after the firing was elicited. Intracellular recordings were made from the hippocampal CA3 pyramidal neurons using a glass microelectrode (electrical resistance: 54–146 M) filled with 3 M KCl. Under the same condition, a depolarizing pulse (0.5-nA intensity, 120-ms duration) was applied to the cell through the recording electrode (filled with 3 M KCl) to induce repetitive firing. The Ca2+ spike was induced by applying depolarizing pulse (0.5-nA intensity, 120-ms duration) in the presence of 1 M tetrodotoxin (TTX) and 10 mM tetraethylammonium (TEA). LEV dissolved in ACSF at a concentration of 10 M to 1 mM and sodium phenytoin (PHT, Dainippon Sumitomo Co. Ltd., Osaka, Japan) of 100 M circulated the perfusion system and bathed the slice preparations for 5 min. Recordings were performed immediately before drug-application to the disappearance of its effect after washing out LEV or PHT from the bath, or to 60 min after application if the value did not recover after LEV-washout. The protocols for animal treatment were received prior approval from our institutional review board: animals were treated in accordance with the guideline for animal experiments stipulated by the Ministry of Education, Culture, Sports, Science and Technology in Japan, and all efforts were made to minimize animal suffering. 2.3. Recording and analysis The responses elicited by MFS and depolarizing pulses into the cell were displayed on a digital oscilloscope (VC-10, Nihon Kohden, Tokyo, Japan) after amplification (amplifier MEZ-8201, Nihon Kohden), and stored on a personal computer (Power Macintosh G4, Apple Inc., CA, USA) using the PowerLab System (AD Instruments Pty Ltd., NSW, Australia). In addition, the responses were continuously recorded on a thermal array recorder (RTA-1100, Nihon Kohden). The differences between pre- and post-treatment with LEV were statistically analyzed by the oneway ANOVA followed by the Fischer test.
3. Results The data were obtained from hippocampal CA3 neurons with a resting membrane potential greater than −45 mV. They were 35 neurons obtained from 27 mature SERs and 9 neurons from 8 normal Wistar rats. The CA3 neurons of SERs were classified into three groups (G) as previously described [10]. Briefly, G1 and G2 (G1/2) neurons showed depolarization shifts lasting for more than 100 ms and 50–100 ms, respectively. The shifts were accompanied by a single MFS-induced repetitive firing (Fig. 1a and Table 1). In contrast, group 3 (G3) neurons lacked the depolarization shift and fired only one spike upon MFS (Fig. 2a). We recorded a larger number of MFSevoked G3 neurons to evaluate the response to LEV in this study, because these neurons exhibited a certain characteristic response to LEV. As the responses of G1/2 neurons to LEV were typical and consistent, they were extracted from previous findings [7,8].
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3.1. Effects of LEV on depolarization shift with repetitive firing We examined the effects of LEV on 10 SER hippocampal CA3 neurons (G 1/2) with MFS-induced depolarization shifts and repetitive firing. The mean duration of the depolarization shift and the number of spikes induced by a single MFS were 68.2 ± 5.5 ms and 5.2 ± 0.8 (n = 10), respectively. In 4 G1/2 neurons, we recorded the effects of LEV at two concentrations in the same neuron after complete recovery with LEV washout. Resting membrane potential did not show significant change throughout LEV application. LEV (10–100 M) concentration-dependently inhibited the MFS-induced depolarization shift and repetitive firing significantly (P < 0.01) (Fig. 1a and Table 1). The inhibitory effect appeared within 10 min and persisted more than 30 min after LEV washout. LEV at a concentration of 1 mM blocked the residual first MFS-evoked action potential in 3 of 4 neurons (the potential remained unaffected in the remaining neuron). However, neither 10 nor 100 M LEV blocked the first spike in any of the 5 G1/2 neurons tested. LEV at 100 M and 1 mM also inhibited after-hyperpolarization following the depolarization shift with repetitive firing in a concentration-dependent manner (Fig. 1b). In G3 neurons, dos-dependent inhibition of response by LEV was obvious: viz., 10 M, 100 M and 1 mM of LEV respectively inhibited of the spike observed in 1 of 5, 4 of 7, and all 6 (P < 0.01) neurons (Fig. 2a and Table 1). In contrast to SER neurons, the single MFS-evoked action potential elicited remained unaffected even with the higher LEV concentration (1 mM) in any of the 3 neurons of normal Wistar rats (Fig. 2b and Table 1). Effect of PHT was examined in 3 G1/2 neurons in SER and 3 neurons in Wistar rats. PHT of 100 M completely suppressed the MFS-induced depolarization shift (57.9 ± 2.6 ms) and repetitive firing (8.0 ± 0.7) including the first spike in all 3 SER G1/2 neurons. The inhibitory effect of PHT was soon disappeared after washout. PHT (100 M) did not affect the MFS-evoked action potential in any CA3 neurons of non-epileptic Wistar rats (Fig. 3). 3.2. Effects of LEV on depolarizing pulse-induced repetitive firing The intracellular application of a depolarizing pulse with a 120ms duration induced repetitive firing in 26 SER and 9 Wistar rat neurons. G1/2 neurons elicited 3.3 ± 0.5 spikes by the depolarizing pulse, while G3 neurons of SER and Wistar rats indicated 2.1 ± 0.3 and 2.1 ± 0.3 spikes, respectively. The number of spikes induced by the depolarizing pulse was significantly higher in G1/2 than in G3 neurons and Wistar rats. Although repetitive firing in 14 SER CA3 neurons of all groups of neurons exposed to LEV was not affected at 10 M; spikes in all G1/2 neurons were markedly (P < 0.05) suppressed at 100 M (Fig. 4 and Table 2). Increasing the concentrations up to 1 mM resulted in a significant (P < 0.05) reduction of the repetitive firing in G1/2 neurons, while there were no significant differences in the inhibitory effect between both doses. These effects appeared within 10 min and decayed within 10-min after LEV washout. The repetitive firing of any G3 neurons was not affected by LEV up to 1 mM. In the control Wistar rats, the single MFS-evoked spike remained unaffected in any 3 of G1/2 or G3 neurons by LEV at 1 mM (Table 2). 3.3. Effects of LEV on Ca2+ spikes Ca2+ spikes were induced by the intracellular application of a depolarizing pulse lasting 120 ms under the blockade of Na+ and K+ channels by TTX and TEA, respectively. The effects of LEV on Ca2+ spikes were examined in 4 G1/2 neurons and in 3 G3 neurons of SER. Times required to reach the peak of Ca2+ spikes were 44.4 ± 12.1 and 46.0 ± 9.7 ms, with maximum spike voltages of 65.5 ± 8.3 and 60.1 ± 2.1 mV were, respectively in G1/2 and G3 There were no significant differences between the neurons with and without
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Control
LEV 100µM
Wash out
20mV
Stimulation 50ms
20mV 1s
Fig. 1. Inhibitions of mossy fiber stimulation (MFS)-evoked depolarization shift with repetitive firing (a) and after-hyperpolarization (b) in a hippocampal CA3 group 2 neuron of a spontaneously epileptic rats (SER) by levetiracetam (LEV, 100 M). Arrows indicate the MFS initiations. Table 1 Effect of levetiracetam on the repetitive firing and depolarization shift induced by mossy fiber stimulation. All statistical values are P < 0.01. Rats
LEV 10 M
Type of neurons Before During Before During Before During Before During
No. of spikes Groups 1 & 2 Depo shift (ms) SER No. of spikes Group 3 Depo shift (ms)
4.67 ± 2.33 ± 82.77 ± 46.73 ± 1.00 ± 0.60 ± – –
2.04 (n = 3) 0.82 (n = 3) 15.12 (n = 3) 29.00 (n = 3) 0.00 (n = 5) 0.27 (n = 5)
LEV 100 M 4.33 ± 1.00 ± 67.43 ± 0.00 ± 1.00 ± 0.43 ± – –
1.63 (n = 3) 0.00** (n = 3) 11.99 (n = 3) 0.00** (n = 3) 0.00 (n = 7) 0.22** (n = 7)
Depo shift (ms)
1.09 (n = 4) 0.29** (n = 4) 6.64 (n = 4) 0.00** (n = 4) 0.00 (n = 6) 0.00** (n = 6)
1.00 ± 0.00 (n = 3) 1.00 ± 0.00 (n = 3) – –
Before During Before During
No. of spikes Wistar
LEV 1 mM 4.25 ± 0.25 ± 71.01 ± 0.00 ± 1.00 ± 0.00 ± – –
Values are the mean ± S.E.M. n, the number of neurons tested; LEV, levetiracetam; Depo shift, depolarizing shift. ** P < 0.01 as compared with values before drug application.
Control
LEV 1mM
Wash out
20mV
Stimulation 50ms
Control
LEV 1mM
Wash out
Fig. 2. LEV (1 mM) effects on the MFS-evoked action potential in a hippocampal CA3 group 3 neuron of SER (a) and that of a non-epileptic Wistar rat (b). Arrows indicate MFS initiations (refer to Fig. 1 for abbreviations).
R. Hanaya et al. / Brain Research Bulletin 86 (2011) 334–339
Control
PHT 100µM
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Wash out
20mV
Stimulation 50ms
Control
PHT 100µM
Wash out
Fig. 3. Phenytoin (PHT, 100 M) effects on the MFS-evoked action potential in a hippocampal CA3 group 2 neuron of SER (a) and that of a non-epileptic Wistar rat (b). Arrows indicate MFS initiations (refer to Fig. 1 for abbreviations).
Control
LEV 100µM
Control
LEV 100µM
Wash out
0.5nA
Wash out
20mV 50ms
0.5nA
Fig. 4. LEV (100 M) induced inhibition on sodium spikes induced by intracellularly applied depolarization pulse (0.5-nA intensity, 120-ms duration) in a group 1 (a) or 3 (b) CA3 neuron of SER (refer to Fig. 1 for abbreviations).
Table 2 Effect of levetiracetam on sodium spikes. Rats
No. of neurons
No. of spikes LEV 10 M
Groups 1 & 2 SER Group 3
Wistar
Before During Before During
3.33 3.00 1.80 1.80
Before During
Values are the mean ± S.E.M. n, the number of neurons tested; LEV, levetiracetam. * P < 0.05 as compared with values before drug application.
± ± ± ±
1.08 (n = 3) 1.22 (n = 3) 0.74 (n = 5) 0.65 (n = 5)
LEV 100 M 3.67 2.00 2.28 2.00
± ± ± ±
1.08 (n = 3) 1.41* (n = 3) 0.45 (n = 7) 0.47 (n = 7)
LEV 1 mM 3.00 1.33 2.20 2.20
± ± ± ±
1.22 (n = 3) 1.63* (n = 3) 0.65 (n = 5) 0.65 (n = 5)
2.33 ± 0.78 (n = 3) 2.33 ± 1.08 (n = 3)
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Control
LEV 100µM
Control
LEV 100µM
Wash out
0.5nA
Wash out
20mV
0.5nA
50ms
Fig. 5. LEV (100 M) did not affect the Ca2+ spikes induced by an intracellularly applied depolarization pulse (0.5-nA intensity, 120-ms duration) in a group 1 (a) or 3 (b) CA3 neuron of SER in the presence of tetrodotoxin (1 M) and tetraethylammonium (10 mM) (refer to Fig. 1 for abbreviations).
MFS-induced abnormal firing. LEV of either 100 M or 1 mM did not affect the amplitude of the Ca2+ spikes in any neuron of the 3 groups (Fig. 5). 4. Discussion The inhibitory effects of LEV continued for over 30 min after LEV washout from the bath in this study. The antiepileptic effects of LEV on SER were relatively delayed and displayed time-related potency, with long-lasting action after a single-bolus drug injection [12,35]. These findings suggest that receptor binding and channel modulation are not involved in the primary mechanism of LEV action, although SV2A serves as a binding site for the antiepileptic drug levetiracetam [23]. LEV reduces GABA transporter (GTRAP3-18) [31], and induces a loss of SV2A to impede action potentialdependent GABAergic neurotransmission [23]. SV2A is decreased during epileptogenesis and chronic epilepsy [32]. These results indicate that LEV enhances GABAergic inhibition. In the present study, LEV inhibited the depolarization shift and repetitive firing in a concentration-dependent manner (Table 1). LEV-enhanced GABAergic inhibition could be an important mechanism to suppress abnormal excitability of CA3 neurons in SER. LEV at 10 M inhibited MFS-evoked depolarization shift and repetitive firing, and this dose is lower than the blood concentration effective in inhibiting epileptic seizures in humans [14]. In contrast, LEV at a much higher concentration (1 mM) did not affect MFS-evoked spike generation in CA3 neurons of normal Wistar rats. SER hippocampus has a lower density of benzodiazepine receptors with normal affinity [29]. GABAA receptor-mediated inhibition is reduced in the epileptogenic hippocampus [11]. Tremor rats, one of the parent strains of SER, exhibit a higher sensitivity to GABA in the hippocampus [16], thus indicating that the SER hippocampus also expresses a higher sensitivity to GABA. In contrast, even in SER G3 CA3 neurons, which displayed no abnormal excitability, MFS-evoked spike generation was inhibited by LEV (10 M to 1 mM). In the SER, there were no genetic changes in the glutamate receptor, or the related cation channels in a linkage study of the position of the glutamate receptor gene with epileptic seizures [17,19]. Neither the ionotropic glutamate receptor nor glutamate
synthesis is affected by LEV [9], although LEV increases glutamate transporter (EAAC-1) levels, and reduces glutamate-induced excitotoxicity could cumulatively have contributed to its antiepileptic action [31]. The MFS-evoked repetitive firing in SER CA3 neuron is thought to reflect the elevation of glutamatergic action [10]. In addition to enhancement of the GABAergic inhibition, LEV might have contributed to the suppression of abnormal firing in SER CA3 neurons via the inhibition of glutamatergic transmission. These effects would therefore explain that LEV suppressed the first spike of G3 CA3 neuron without abnormal firing. Prophylactic treatment of LEV inhibits hippocampal sclerosis in SER [30], and the effects of LEV on synaptic transmission would have contributed to neuroprotection in SER hippocampus; a mechanism similarly displayed by other antiepileptic drugs with a neuroprotective effect [13]. LEV-induced inhibition appeared within 5 min after bath application. In this experimental system, the latency of inhibitory expression of LEV was similar to that of the other channel-acting antiepileptic drugs examined. The early-onset inhibitory effect of LEV on MFS-evoked CA3 excitability suggests that LEV also elicits an acute and direct action relevant to receptors or channels. LEV influences the N-type Ca2+ channel [22]. LEV appears to have no direct effects on the L-type Ca2+ channel, since the drug does not affect the Ca2+ spike amplitude sensitive to an L-type Ca2+ antagonist (nicardipine) in SER CA3 neurons [25]. Further study of the LEV are required for the effect of LEV on Ca2+ spike because the shape of the Ca2+ spike is slightly different from the G1/2 and G3. Our present findings demonstrate that LEV (100 M, 1 mM) specifically reduced the firings induced by depolarizing pulse in SER G1/2 CA3 neurons without affecting similar firings in SER G3 neurons and Wistar rat neurons. LEV did not have a direct effect on the Na+ channel [37], although brivaracetam, a pyrrolidone derivative with a one-log-unit higher affinity than LEV to SV2A, inhibits Na+ current in rat cortical neurons in cultures [36]. At the same time, the SER hippocampus display abnormality in the voltage-gate sodium channel [6]. PHT inhibited the MFS-evoked abnormal firing including the first spike in SER CA3 neurons nevertheless the first spike elicited by MFS was not affected by PHT in Wistar rats. PHT shows the antiepileptic effect by blocking the Na+ channel. These indicate
R. Hanaya et al. / Brain Research Bulletin 86 (2011) 334–339
that LEV with a therapeutic dose may affect those hyperactive Na+ channels which cause cellular excitability. We divided the SER CA3 neurons into 3 groups based on the response to MFS. The Ca2+ channel is hypersensitive in all CA3 dissociated neurons of SER [34], albeit one-thirds of CA3 neurons do not exhibit MFS-evoked abnormal excitability. The MFS-evoked abnormal firing in SER CA3 neurons presumably reflect the Na+ channel activity in addition to glutamatergic action [10]. Our results support that hyperactivity of the Na+ channels in G1/2 neurons was one of the factors contributing to the MFS-evoked abnormal response. In conclusion, LEV inhibited MFS-evoked abnormal excitability in SER hippocampal CA3 neurons mainly by modulation of synaptic transmission. Our results suggest that LEV at the therapeutic concentration may contribute to the suppression mechanism of epileptic seizures without affecting normal neuronal transmission. Conflict of interest The authors have no conflict of interest. Acknowledgements Levetiracetam was kindly provided by UCB, Inc., Belgium. The authors are very grateful to Dr. H. Klitgaard for critical reading of the manuscript. References [1] T. Amano, H. Amano, H. Matsubayashi, K. Ishihara, T. Serikawa, M. Sasa, Enhanced Ca(2+) influx with mossy fiber stimulation in hippocampal CA3 neurons of spontaneously epileptic rats, Brain Res. 910 (2001) 199–203. [2] K.M. Crowder, J.M. Gunther, T.A. Jones, B.D. Hale, H.Z. Zhang, M.R. Peterson, R.H. Scheller, C. Chavkin, S.M. Bajjalieh, Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A), Proc. Natl. Acad. Sci. U.S.A. 96 (1996) 15268–15273. [3] C.M. Fraser, G.J. Sills, E. Butler, G.G. Thompson, K. Lindsay, R. Duncan, A. Howatson, M.J. Brodie, Effects of valproate, vigabatrin and tiagabin on GABA uptake into human astrocytes cultured from fetal and adult brain tissue, Epileptic. Disord. 1 (1999) 153–157. [4] P. Genton, B. Vleymen, Piracetam and levetiracetam: close structural similarities but different pharmacological and clinical profiles, Epileptic. Disord. 2 (2000) 99–105. [5] A.J. Gower, E. Hirsch, A. Boehrer, M. Noyer, C. Marescaux, Effects of levetiracetam, a novel antiepileptic drug, on convulsant activity in two genetic rat models of epilepsy, Epilepsy Res. 22 (1995) 207–213. [6] F. Guo, N. Yu, J.Q. Cai, T. Quinn, Z.H. Zong, Y.J. Zeng, L.Y. Hao, Voltage-gated sodium channel Nav1.1, Nav1. 3 and beta1 subunit were up-regulated in the hippocampus of spontaneously epileptic rat, Brain Res. Bull. 75 (2008) 179–187. [7] R. Hanaya, M. Sasa, Y. Kiura, T. Serikawa, K. Kurisu, Effects of vigabatrin on epileptiform abnormal discharges in hippocampal CA3 neurons of spontaneously epileptic rat (SER), Epilepsy Res. 50 (2002) 223–231. [8] R. Hanaya, M. Sasa, H. Ujihara, K. Ishihara, T. Serikawa, K. Iida, T. Akimitsu, K. Arita, K. Kurisu, Suppression by topiramate of epileptic burst discharges in hippocampal CA3 neurons of spontaneously epileptic rat in vitro, Brain Res. 789 (1998) 274–282. [9] G. Hans, L. Nguyen, V. Rocher, S. Belachew, G. Moonen, A. Matagne, H. Klitgaard, Levetiracetam: no relevant effect on ionotropic excitatory glutamate receptors, Epilepsia 41 (Suppl. 7) (2000) 35. [10] K. Ishihara, M. Sasa, T. Momiyama, H. Ujihara, J. Nakamura, T. Serikawa, J. Yamada, S. Takaori, Abnormal excitability of hippocampal CA3 pyramidal neurons of spontaneously epileptic rats (SER), a double mutant, Exp. Neurol. 119 (1993) 287–290. [11] M. Isokawa, Modulation of GABAA receptor-mediated inhibition by postsynaptic calcium in epileptic hippocampal neurons, Brain Res. 810 (1998) 241–250. [12] C. Ji-qun, K. Ishihara, T. Nagayama, T. Serikawa, M. Sasa, Long-lasting antiepileptic effects of levetiracetam against epileptic seizures in the spontaneously epileptic rat (SER): differentiation of levetiracetam from conventional
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Mehraein, A.P. Anzil, F. Deerberg, A new rat mutant with detective overhairs and spongy degeneration of the central nervus system: clinical pathological studies, Lab. Anim. Sci. 32 (1982) 70–73. [27] M. Sasa, Y. Ohno, H. Ujihara, Y. Fujita, M. Yoshimura, S. Takaori, T. Serikawa, J. Yamada, Effects of antiepileptic drugs on absence-like and tonic seizures in the spontaneously epileptic rat, a double mutant rat, Epilepsia 29 (1988) 505–513. [28] T. Serikawa, J. Yamada, Epileptic seizures in rats homozygous for two mutations, zitter and tremor, J. Hered. 77 (1986) 441–444. [29] Y. Shirasaka, M. Ito, I. Mitsuyoshi, H. Mikawa, T. Serikawa, J. Yamada, Benzodiazepine receptor binding in the spontaneously epileptic rat and its parent strains, Exp. Neurol. 113 (1991) 255–259. [30] S. Sugata, R. Hanaya, K. Kumafuji, M. Tokudome, T. Serikawa, K. Kurisu, K. Arita, M. Sasa, Neuroprotective effect of levetiracetam on hippocampal sclerosis-like change in spontaneously epileptic rats, Brain Res. Bull. 86 (2001) 36–41. [31] Y. Ueda, T. Doi, K. Nagatomo, J. Tokumaru, M. Takaki, L.J. Willmore, Effect of levetiracetam on molecular regulation of hippocampal glutamate and GABA transporters in rats with chronic seizures induced by amygdalar FeCl3 injection, Brain Res. 1151 (2007) 55–61. [32] E.A. van Vliet, E. Aronica, S. Redeker, K. Boer, J.A. Gorter, Decreased expression of synaptic vesicle protein 2A, the binding site for levetiracetam, during epileptogenesis and chronic epilepsy, Epilepsia 50 (2009) 422–433. [33] J. Yamada, T. Serikawa, J. Ishiko, T. Inui, H. Takada, Y. Kawai, A. Okaniwa, Rats with congenital tremor and curled whisker and hair, Exp. Anim. 34 (1985) 183–188. [34] H.D. Yan, K. Ishihara, R. Hanaya, K. Kurisu, T. Serikawa, M. Sasa, Voltagedependent calcium channel abnormalities in hippocampal CA3 neurons of spontaneously epileptic rats, Epilepsia 48 (2007) 758–764. [35] H.D. Yan, C. Ji-qun, K. Ishihara, T. Nagayama, T. Serikawa, M. Sasa, Separation of antiepileptogenic and antiseizure effects of levetiracetam in the spontaneously epileptic rat (SER), Epilepsia 46 (2005) 1170–1177. [36] C. Zona, M. Pieri, I. Carunchio, L. Curcio, H. Klitgaard, D.G. Margineanu, Brivaracetam (ucb 34714) inhibits Na(+) current in rat cortical neurons in culture, Epilepsy Res. 88 (2010) 46–54. [37] C. Zona, I. Niespodziany, C. Marchetti, H. Klitgaard, G. Bernardi, D.G. Margineanu, Levetiracetam does not modulate neuronal voltage-gated Na+ and T-type Ca2+ currents, Seizure 10 (2001) 279–286.
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Research report
Modulation of abnormal synaptic transmission in hippocampal CA3 neurons of spontaneously epileptic rats (SERs) by levetiracetam Ryosuke Hanaya a,∗ , Yoshihiro Kiura b , Tadao Serikawa c , Kaoru Kurisu b , Kazunori Arita a , Masashi Sasa d a
Department of Neurosurgery, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima 890-8544, Japan Department of Neurosurgery, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan d Nagisa Clinic, Hirakata 573-1183, Japan b c
a r t i c l e
i n f o
Article history: Received 4 August 2011 Received in revised form 17 September 2011 Accepted 19 September 2011 Available online 25 September 2011 Keywords: Levetiracetam Spontaneously epileptic rat (SER) Hippocampal CA3 neurons Depolarization shift GABAergic transmission Sodium channel
a b s t r a c t Levetiracetam (LEV) inhibits partial refractory epilepsy in human, and both convulsive and absence-like seizures in the spontaneously epileptic rat (SER). Two-thirds of hippocampal CA3 neurons in SER show a long-lasting depolarization shift, with accompanying repetitive firing upon mossy fiber stimulation. This abnormal excitability is probably attributable to abnormalities in the L-type Ca2+ channels. We performed electrophysiological studies to elucidate the mechanism underlying the antiepileptic effects of LEV via intracellular recording from the hippocampal CA3 neurons in slice preparations of SER and non-epileptic Wistar rats. LEV (100 M) inhibited the depolarization shift with repetitive firing by mossy fiber stimulation (MFS), without affecting the first spike in SER CA3 neurons. At a higher dose (1 mM), LEV suppressed the first spike in all SER neurons (including the CA3 neurons which showed only a single action potential by MFS), while the single action potential of Wistar rat CA3 neurons remained unaffected. SER CA3 neurons with MFS-induced abnormal firing exhibited a higher number of repetitive spikes when a depolarization pulse was applied in the SER CA3 neurons. LEV (100 M, 1 mM) reduced the repetitive firing induced by a depolarization pulse applied without affecting Ca2+ spike in SER neurons. LEV is known not to bind glutamate and gamma-aminobutyric acid (GABA) receptors. These findings suggest that the therapeutic concentration of LEV inhibits abnormal firing of the CA3 neurons by modulating abnormal synaptic transmission and abnormal Na+ channels in SER. © 2011 Elsevier Inc. All rights reserved.
1. Introduction Levetiracetam (LEV), a pyrrolidone derivative structurally related to piracetam [4], is effective to inhibit the seizures in kindled and genetic animals [5,18,21] and for the treatment of both primary generalized and partial epilepsy in humans [15,20]. LEV, interestingly, has no remarkable effects on acute maximal electroshock or pentylentetrazole-induced seizures [18]. Unlike the conventional antiepileptic drugs, LEV does not directly affect either Na+ channels, T-type Ca2+ channels [37], glutamate receptors [9], or gamma-aminobutyric acid (GABA) systems [3,24]. LEV has been found to inhibit N-type Ca2+ channels [22] and to specifically bind to synaptic vesicle protein 2A (SV2A) [23], which is presumably involved in regulating neurotransmitter release [2].
∗ Corresponding author at: Department of Neurosurgery, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan. Tel.: +81 99 275 5375; fax: +81 99 265 4041. E-mail address: [email protected] (R. Hanaya). 0361-9230/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.brainresbull.2011.09.015
Our previous studies have demonstrated that LEV is effective in inhibiting both tonic convulsion and absence-like seizures in spontaneously epileptic rats (SERs: zi/zi, tm/tm) [12,35]. SER is a double mutant obtained by mating heterozygous tremor rats (tm/+), a mutant found in an inbred colony of Kyoto-Wistar rats [33], with homozygous zitter rats (zi/zi) originated from a Sprague-Dawley colony [26]. After 6 weeks of age, SERs spontaneously begin to show tonic convulsion and absence-like seizures characterized by respectively low voltage fast waves and 5–7 Hz spike-wave-like seizures in cortical and hippocampal EEG [27,28]. The profiles of conventional antiepileptic drugs for SER parallel those in human epilepsy [28]. A single stimulation of the mossy fibers (MF) induces a long-lasting (60–200 ms) depolarization shift, accompanied by repetitive firing and slow after-hyperpolarization in two thirds of the hippocampal CA3 neurons in SER [10]. Enhancement of intracellular Ca2+ levels with the stimulation of MF has been observed in the hippocampal CA3 neurons of SER, compared with normal rats [1]. This abnormal excitability would induce abnormalities in the L-type Ca2+ channels [25,34]. The MF-hippocampal CA3 neuron system of SER is one of the useful targets for elucidating the
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mechanism underlying the antiepileptic action of drugs [8,7]. We performed electrophysiological studies to elucidate the antiepileptic mechanism of LEV using SER hippocampal slices. 2. Materials and methods 2.1. Experimental animals SERs were bred in a specific pathogen-free barriered facility in the Institute of Laboratory Animals, Kyoto University, and were kept individually in shoebox type cages on laminar flow shelves in a conventional room throughout the experiment. Animal feed pellets (F-2, Funabashi Farm, Chiba, Japan) and drinking water were given to the animals ad libitum. The room temperature and relative humidity were maintained at 22–26 ◦ C and 50–65%, respectively. We used 29 mature (age: 10–16 weeks) SERs of either sex that exhibited convulsive seizures, and 10 age-matched Wistar rats, one of the parent strains of SERs, were used as controls. 2.2. Intracellular recording in hippocampal slices After deep anesthesia with ether, the brain of rodents was removed and rapidly placed in cold oxygenated Ca2+ -free medium (in mM: 113 NaCl, 3 KCl, 1 NaH2 PO4 , 25 NaHCO3 , 5 MgCl2 , 11 glucose), and hippocampal slices were cut at a thickness of 400 m with a microslicer (DTK-1000, Dosaka EM, Kyoto, Japan). After incubation for 1–2 h in artificial cerebrospinal fluid (ACSF; in mM: 113 NaCl, 3 KCl, 1 NaH2 PO4 , 25 NaHCO3 , 2 CaCl2 , 1 MgCl2 , 11 glucose; pH 7.2) at 34 ◦ C, the slices were placed into the recording chamber (volume: 0.7 cm3 ). ACSF continuously bubbled with a mixture of 95% O2 and 5% CO2 was perfused over the slice at a rate of 1.5–2.0 ml/min at room temperature. A bipolar stimulating electrode was placed in the granule cell layer of the dentate gyrus to provide a stimulus (0.1 ms-duration and 10–25 V) to the MF every 5 s. The MF stimulation (MFS) raised the threshold by 0.5 V, and held the intensity at 0.5 V higher after the firing was elicited. Intracellular recordings were made from the hippocampal CA3 pyramidal neurons using a glass microelectrode (electrical resistance: 54–146 M) filled with 3 M KCl. Under the same condition, a depolarizing pulse (0.5-nA intensity, 120-ms duration) was applied to the cell through the recording electrode (filled with 3 M KCl) to induce repetitive firing. The Ca2+ spike was induced by applying depolarizing pulse (0.5-nA intensity, 120-ms duration) in the presence of 1 M tetrodotoxin (TTX) and 10 mM tetraethylammonium (TEA). LEV dissolved in ACSF at a concentration of 10 M to 1 mM and sodium phenytoin (PHT, Dainippon Sumitomo Co. Ltd., Osaka, Japan) of 100 M circulated the perfusion system and bathed the slice preparations for 5 min. Recordings were performed immediately before drug-application to the disappearance of its effect after washing out LEV or PHT from the bath, or to 60 min after application if the value did not recover after LEV-washout. The protocols for animal treatment were received prior approval from our institutional review board: animals were treated in accordance with the guideline for animal experiments stipulated by the Ministry of Education, Culture, Sports, Science and Technology in Japan, and all efforts were made to minimize animal suffering. 2.3. Recording and analysis The responses elicited by MFS and depolarizing pulses into the cell were displayed on a digital oscilloscope (VC-10, Nihon Kohden, Tokyo, Japan) after amplification (amplifier MEZ-8201, Nihon Kohden), and stored on a personal computer (Power Macintosh G4, Apple Inc., CA, USA) using the PowerLab System (AD Instruments Pty Ltd., NSW, Australia). In addition, the responses were continuously recorded on a thermal array recorder (RTA-1100, Nihon Kohden). The differences between pre- and post-treatment with LEV were statistically analyzed by the oneway ANOVA followed by the Fischer test.
3. Results The data were obtained from hippocampal CA3 neurons with a resting membrane potential greater than −45 mV. They were 35 neurons obtained from 27 mature SERs and 9 neurons from 8 normal Wistar rats. The CA3 neurons of SERs were classified into three groups (G) as previously described [10]. Briefly, G1 and G2 (G1/2) neurons showed depolarization shifts lasting for more than 100 ms and 50–100 ms, respectively. The shifts were accompanied by a single MFS-induced repetitive firing (Fig. 1a and Table 1). In contrast, group 3 (G3) neurons lacked the depolarization shift and fired only one spike upon MFS (Fig. 2a). We recorded a larger number of MFSevoked G3 neurons to evaluate the response to LEV in this study, because these neurons exhibited a certain characteristic response to LEV. As the responses of G1/2 neurons to LEV were typical and consistent, they were extracted from previous findings [7,8].
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3.1. Effects of LEV on depolarization shift with repetitive firing We examined the effects of LEV on 10 SER hippocampal CA3 neurons (G 1/2) with MFS-induced depolarization shifts and repetitive firing. The mean duration of the depolarization shift and the number of spikes induced by a single MFS were 68.2 ± 5.5 ms and 5.2 ± 0.8 (n = 10), respectively. In 4 G1/2 neurons, we recorded the effects of LEV at two concentrations in the same neuron after complete recovery with LEV washout. Resting membrane potential did not show significant change throughout LEV application. LEV (10–100 M) concentration-dependently inhibited the MFS-induced depolarization shift and repetitive firing significantly (P < 0.01) (Fig. 1a and Table 1). The inhibitory effect appeared within 10 min and persisted more than 30 min after LEV washout. LEV at a concentration of 1 mM blocked the residual first MFS-evoked action potential in 3 of 4 neurons (the potential remained unaffected in the remaining neuron). However, neither 10 nor 100 M LEV blocked the first spike in any of the 5 G1/2 neurons tested. LEV at 100 M and 1 mM also inhibited after-hyperpolarization following the depolarization shift with repetitive firing in a concentration-dependent manner (Fig. 1b). In G3 neurons, dos-dependent inhibition of response by LEV was obvious: viz., 10 M, 100 M and 1 mM of LEV respectively inhibited of the spike observed in 1 of 5, 4 of 7, and all 6 (P < 0.01) neurons (Fig. 2a and Table 1). In contrast to SER neurons, the single MFS-evoked action potential elicited remained unaffected even with the higher LEV concentration (1 mM) in any of the 3 neurons of normal Wistar rats (Fig. 2b and Table 1). Effect of PHT was examined in 3 G1/2 neurons in SER and 3 neurons in Wistar rats. PHT of 100 M completely suppressed the MFS-induced depolarization shift (57.9 ± 2.6 ms) and repetitive firing (8.0 ± 0.7) including the first spike in all 3 SER G1/2 neurons. The inhibitory effect of PHT was soon disappeared after washout. PHT (100 M) did not affect the MFS-evoked action potential in any CA3 neurons of non-epileptic Wistar rats (Fig. 3). 3.2. Effects of LEV on depolarizing pulse-induced repetitive firing The intracellular application of a depolarizing pulse with a 120ms duration induced repetitive firing in 26 SER and 9 Wistar rat neurons. G1/2 neurons elicited 3.3 ± 0.5 spikes by the depolarizing pulse, while G3 neurons of SER and Wistar rats indicated 2.1 ± 0.3 and 2.1 ± 0.3 spikes, respectively. The number of spikes induced by the depolarizing pulse was significantly higher in G1/2 than in G3 neurons and Wistar rats. Although repetitive firing in 14 SER CA3 neurons of all groups of neurons exposed to LEV was not affected at 10 M; spikes in all G1/2 neurons were markedly (P < 0.05) suppressed at 100 M (Fig. 4 and Table 2). Increasing the concentrations up to 1 mM resulted in a significant (P < 0.05) reduction of the repetitive firing in G1/2 neurons, while there were no significant differences in the inhibitory effect between both doses. These effects appeared within 10 min and decayed within 10-min after LEV washout. The repetitive firing of any G3 neurons was not affected by LEV up to 1 mM. In the control Wistar rats, the single MFS-evoked spike remained unaffected in any 3 of G1/2 or G3 neurons by LEV at 1 mM (Table 2). 3.3. Effects of LEV on Ca2+ spikes Ca2+ spikes were induced by the intracellular application of a depolarizing pulse lasting 120 ms under the blockade of Na+ and K+ channels by TTX and TEA, respectively. The effects of LEV on Ca2+ spikes were examined in 4 G1/2 neurons and in 3 G3 neurons of SER. Times required to reach the peak of Ca2+ spikes were 44.4 ± 12.1 and 46.0 ± 9.7 ms, with maximum spike voltages of 65.5 ± 8.3 and 60.1 ± 2.1 mV were, respectively in G1/2 and G3 There were no significant differences between the neurons with and without
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Control
LEV 100µM
Wash out
20mV
Stimulation 50ms
20mV 1s
Fig. 1. Inhibitions of mossy fiber stimulation (MFS)-evoked depolarization shift with repetitive firing (a) and after-hyperpolarization (b) in a hippocampal CA3 group 2 neuron of a spontaneously epileptic rats (SER) by levetiracetam (LEV, 100 M). Arrows indicate the MFS initiations. Table 1 Effect of levetiracetam on the repetitive firing and depolarization shift induced by mossy fiber stimulation. All statistical values are P < 0.01. Rats
LEV 10 M
Type of neurons Before During Before During Before During Before During
No. of spikes Groups 1 & 2 Depo shift (ms) SER No. of spikes Group 3 Depo shift (ms)
4.67 ± 2.33 ± 82.77 ± 46.73 ± 1.00 ± 0.60 ± – –
2.04 (n = 3) 0.82 (n = 3) 15.12 (n = 3) 29.00 (n = 3) 0.00 (n = 5) 0.27 (n = 5)
LEV 100 M 4.33 ± 1.00 ± 67.43 ± 0.00 ± 1.00 ± 0.43 ± – –
1.63 (n = 3) 0.00** (n = 3) 11.99 (n = 3) 0.00** (n = 3) 0.00 (n = 7) 0.22** (n = 7)
Depo shift (ms)
1.09 (n = 4) 0.29** (n = 4) 6.64 (n = 4) 0.00** (n = 4) 0.00 (n = 6) 0.00** (n = 6)
1.00 ± 0.00 (n = 3) 1.00 ± 0.00 (n = 3) – –
Before During Before During
No. of spikes Wistar
LEV 1 mM 4.25 ± 0.25 ± 71.01 ± 0.00 ± 1.00 ± 0.00 ± – –
Values are the mean ± S.E.M. n, the number of neurons tested; LEV, levetiracetam; Depo shift, depolarizing shift. ** P < 0.01 as compared with values before drug application.
Control
LEV 1mM
Wash out
20mV
Stimulation 50ms
Control
LEV 1mM
Wash out
Fig. 2. LEV (1 mM) effects on the MFS-evoked action potential in a hippocampal CA3 group 3 neuron of SER (a) and that of a non-epileptic Wistar rat (b). Arrows indicate MFS initiations (refer to Fig. 1 for abbreviations).
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Control
PHT 100µM
337
Wash out
20mV
Stimulation 50ms
Control
PHT 100µM
Wash out
Fig. 3. Phenytoin (PHT, 100 M) effects on the MFS-evoked action potential in a hippocampal CA3 group 2 neuron of SER (a) and that of a non-epileptic Wistar rat (b). Arrows indicate MFS initiations (refer to Fig. 1 for abbreviations).
Control
LEV 100µM
Control
LEV 100µM
Wash out
0.5nA
Wash out
20mV 50ms
0.5nA
Fig. 4. LEV (100 M) induced inhibition on sodium spikes induced by intracellularly applied depolarization pulse (0.5-nA intensity, 120-ms duration) in a group 1 (a) or 3 (b) CA3 neuron of SER (refer to Fig. 1 for abbreviations).
Table 2 Effect of levetiracetam on sodium spikes. Rats
No. of neurons
No. of spikes LEV 10 M
Groups 1 & 2 SER Group 3
Wistar
Before During Before During
3.33 3.00 1.80 1.80
Before During
Values are the mean ± S.E.M. n, the number of neurons tested; LEV, levetiracetam. * P < 0.05 as compared with values before drug application.
± ± ± ±
1.08 (n = 3) 1.22 (n = 3) 0.74 (n = 5) 0.65 (n = 5)
LEV 100 M 3.67 2.00 2.28 2.00
± ± ± ±
1.08 (n = 3) 1.41* (n = 3) 0.45 (n = 7) 0.47 (n = 7)
LEV 1 mM 3.00 1.33 2.20 2.20
± ± ± ±
1.22 (n = 3) 1.63* (n = 3) 0.65 (n = 5) 0.65 (n = 5)
2.33 ± 0.78 (n = 3) 2.33 ± 1.08 (n = 3)
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Control
LEV 100µM
Control
LEV 100µM
Wash out
0.5nA
Wash out
20mV
0.5nA
50ms
Fig. 5. LEV (100 M) did not affect the Ca2+ spikes induced by an intracellularly applied depolarization pulse (0.5-nA intensity, 120-ms duration) in a group 1 (a) or 3 (b) CA3 neuron of SER in the presence of tetrodotoxin (1 M) and tetraethylammonium (10 mM) (refer to Fig. 1 for abbreviations).
MFS-induced abnormal firing. LEV of either 100 M or 1 mM did not affect the amplitude of the Ca2+ spikes in any neuron of the 3 groups (Fig. 5). 4. Discussion The inhibitory effects of LEV continued for over 30 min after LEV washout from the bath in this study. The antiepileptic effects of LEV on SER were relatively delayed and displayed time-related potency, with long-lasting action after a single-bolus drug injection [12,35]. These findings suggest that receptor binding and channel modulation are not involved in the primary mechanism of LEV action, although SV2A serves as a binding site for the antiepileptic drug levetiracetam [23]. LEV reduces GABA transporter (GTRAP3-18) [31], and induces a loss of SV2A to impede action potentialdependent GABAergic neurotransmission [23]. SV2A is decreased during epileptogenesis and chronic epilepsy [32]. These results indicate that LEV enhances GABAergic inhibition. In the present study, LEV inhibited the depolarization shift and repetitive firing in a concentration-dependent manner (Table 1). LEV-enhanced GABAergic inhibition could be an important mechanism to suppress abnormal excitability of CA3 neurons in SER. LEV at 10 M inhibited MFS-evoked depolarization shift and repetitive firing, and this dose is lower than the blood concentration effective in inhibiting epileptic seizures in humans [14]. In contrast, LEV at a much higher concentration (1 mM) did not affect MFS-evoked spike generation in CA3 neurons of normal Wistar rats. SER hippocampus has a lower density of benzodiazepine receptors with normal affinity [29]. GABAA receptor-mediated inhibition is reduced in the epileptogenic hippocampus [11]. Tremor rats, one of the parent strains of SER, exhibit a higher sensitivity to GABA in the hippocampus [16], thus indicating that the SER hippocampus also expresses a higher sensitivity to GABA. In contrast, even in SER G3 CA3 neurons, which displayed no abnormal excitability, MFS-evoked spike generation was inhibited by LEV (10 M to 1 mM). In the SER, there were no genetic changes in the glutamate receptor, or the related cation channels in a linkage study of the position of the glutamate receptor gene with epileptic seizures [17,19]. Neither the ionotropic glutamate receptor nor glutamate
synthesis is affected by LEV [9], although LEV increases glutamate transporter (EAAC-1) levels, and reduces glutamate-induced excitotoxicity could cumulatively have contributed to its antiepileptic action [31]. The MFS-evoked repetitive firing in SER CA3 neuron is thought to reflect the elevation of glutamatergic action [10]. In addition to enhancement of the GABAergic inhibition, LEV might have contributed to the suppression of abnormal firing in SER CA3 neurons via the inhibition of glutamatergic transmission. These effects would therefore explain that LEV suppressed the first spike of G3 CA3 neuron without abnormal firing. Prophylactic treatment of LEV inhibits hippocampal sclerosis in SER [30], and the effects of LEV on synaptic transmission would have contributed to neuroprotection in SER hippocampus; a mechanism similarly displayed by other antiepileptic drugs with a neuroprotective effect [13]. LEV-induced inhibition appeared within 5 min after bath application. In this experimental system, the latency of inhibitory expression of LEV was similar to that of the other channel-acting antiepileptic drugs examined. The early-onset inhibitory effect of LEV on MFS-evoked CA3 excitability suggests that LEV also elicits an acute and direct action relevant to receptors or channels. LEV influences the N-type Ca2+ channel [22]. LEV appears to have no direct effects on the L-type Ca2+ channel, since the drug does not affect the Ca2+ spike amplitude sensitive to an L-type Ca2+ antagonist (nicardipine) in SER CA3 neurons [25]. Further study of the LEV are required for the effect of LEV on Ca2+ spike because the shape of the Ca2+ spike is slightly different from the G1/2 and G3. Our present findings demonstrate that LEV (100 M, 1 mM) specifically reduced the firings induced by depolarizing pulse in SER G1/2 CA3 neurons without affecting similar firings in SER G3 neurons and Wistar rat neurons. LEV did not have a direct effect on the Na+ channel [37], although brivaracetam, a pyrrolidone derivative with a one-log-unit higher affinity than LEV to SV2A, inhibits Na+ current in rat cortical neurons in cultures [36]. At the same time, the SER hippocampus display abnormality in the voltage-gate sodium channel [6]. PHT inhibited the MFS-evoked abnormal firing including the first spike in SER CA3 neurons nevertheless the first spike elicited by MFS was not affected by PHT in Wistar rats. PHT shows the antiepileptic effect by blocking the Na+ channel. These indicate
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that LEV with a therapeutic dose may affect those hyperactive Na+ channels which cause cellular excitability. We divided the SER CA3 neurons into 3 groups based on the response to MFS. The Ca2+ channel is hypersensitive in all CA3 dissociated neurons of SER [34], albeit one-thirds of CA3 neurons do not exhibit MFS-evoked abnormal excitability. The MFS-evoked abnormal firing in SER CA3 neurons presumably reflect the Na+ channel activity in addition to glutamatergic action [10]. Our results support that hyperactivity of the Na+ channels in G1/2 neurons was one of the factors contributing to the MFS-evoked abnormal response. In conclusion, LEV inhibited MFS-evoked abnormal excitability in SER hippocampal CA3 neurons mainly by modulation of synaptic transmission. Our results suggest that LEV at the therapeutic concentration may contribute to the suppression mechanism of epileptic seizures without affecting normal neuronal transmission. Conflict of interest The authors have no conflict of interest. Acknowledgements Levetiracetam was kindly provided by UCB, Inc., Belgium. The authors are very grateful to Dr. H. Klitgaard for critical reading of the manuscript. References [1] T. Amano, H. Amano, H. Matsubayashi, K. Ishihara, T. Serikawa, M. Sasa, Enhanced Ca(2+) influx with mossy fiber stimulation in hippocampal CA3 neurons of spontaneously epileptic rats, Brain Res. 910 (2001) 199–203. [2] K.M. Crowder, J.M. Gunther, T.A. Jones, B.D. Hale, H.Z. Zhang, M.R. Peterson, R.H. Scheller, C. Chavkin, S.M. Bajjalieh, Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A), Proc. Natl. Acad. Sci. U.S.A. 96 (1996) 15268–15273. [3] C.M. Fraser, G.J. Sills, E. Butler, G.G. Thompson, K. Lindsay, R. Duncan, A. Howatson, M.J. Brodie, Effects of valproate, vigabatrin and tiagabin on GABA uptake into human astrocytes cultured from fetal and adult brain tissue, Epileptic. Disord. 1 (1999) 153–157. [4] P. Genton, B. Vleymen, Piracetam and levetiracetam: close structural similarities but different pharmacological and clinical profiles, Epileptic. Disord. 2 (2000) 99–105. [5] A.J. Gower, E. Hirsch, A. Boehrer, M. Noyer, C. Marescaux, Effects of levetiracetam, a novel antiepileptic drug, on convulsant activity in two genetic rat models of epilepsy, Epilepsy Res. 22 (1995) 207–213. [6] F. Guo, N. Yu, J.Q. Cai, T. Quinn, Z.H. Zong, Y.J. Zeng, L.Y. Hao, Voltage-gated sodium channel Nav1.1, Nav1. 3 and beta1 subunit were up-regulated in the hippocampus of spontaneously epileptic rat, Brain Res. Bull. 75 (2008) 179–187. [7] R. Hanaya, M. Sasa, Y. Kiura, T. Serikawa, K. Kurisu, Effects of vigabatrin on epileptiform abnormal discharges in hippocampal CA3 neurons of spontaneously epileptic rat (SER), Epilepsy Res. 50 (2002) 223–231. [8] R. Hanaya, M. Sasa, H. Ujihara, K. Ishihara, T. Serikawa, K. Iida, T. Akimitsu, K. Arita, K. Kurisu, Suppression by topiramate of epileptic burst discharges in hippocampal CA3 neurons of spontaneously epileptic rat in vitro, Brain Res. 789 (1998) 274–282. [9] G. Hans, L. Nguyen, V. Rocher, S. Belachew, G. Moonen, A. Matagne, H. Klitgaard, Levetiracetam: no relevant effect on ionotropic excitatory glutamate receptors, Epilepsia 41 (Suppl. 7) (2000) 35. [10] K. Ishihara, M. Sasa, T. Momiyama, H. Ujihara, J. Nakamura, T. Serikawa, J. Yamada, S. Takaori, Abnormal excitability of hippocampal CA3 pyramidal neurons of spontaneously epileptic rats (SER), a double mutant, Exp. Neurol. 119 (1993) 287–290. [11] M. Isokawa, Modulation of GABAA receptor-mediated inhibition by postsynaptic calcium in epileptic hippocampal neurons, Brain Res. 810 (1998) 241–250. [12] C. Ji-qun, K. Ishihara, T. Nagayama, T. Serikawa, M. Sasa, Long-lasting antiepileptic effects of levetiracetam against epileptic seizures in the spontaneously epileptic rat (SER): differentiation of levetiracetam from conventional
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